Method for the prevention and/or the biological control of bacterial wilt caused by ralstonia solanacearum, via the use of bacteriophages suitable for this purpose and compositions thereof

ABSTRACT

A method is for prevention and/or biological control of wilt caused by  Ralstonia solanacearum,  by use of suitable bacteriophages. In addition a method uses the structural characterisation, genome sequence and activity of three specific lytic bacteriophages of  R. solanacearum.  Podovirus presents an elevated stability between 4° C. and 30° C. in an aqueous medium in the absence of a host. As a result of the high level of stability, lytic activity, elevated specificity towards  R. solanacearum  and the absence of activity against the microbiota associated with the plants to be protected, bacteriophages are used for the biological control of  R. solanacearum  in river courses and irrigation water, as well as in a method for preventing and/or controlling the wilt produced by the bacteria, in which at least one of the bacteriophages, or combinations thereof, are delivered to the plants and/or the soil in the irrigation water.

TECHNICAL FIELD

The invention relates to biological control of pathogenic organisms tocrop plants. More specifically, the invention relates to a method forthe prevention and/or biological control of bacterial wilt caused byRalstonia solanacearum, to bacteriophages useful therefor and to the useof the aforementioned bacteriophages and of compositions containing themfor biological control of this bacterium.

BACKGROUND OF THE INVENTION

R. solanacearum produces bacterial wilt worldwide in more than 200 plantspecies belonging to more than 50 botanical families, and many of thesespecies susceptible to this pathogen are of agricultural interest. Thereare also many other crops that are colonized by the bacteria, but do notdevelop symptoms, which are considered tolerant crops. This bacteriumespecially attacks staple crops such as potatoes in more than eightycountries, with losses exceeding 950 million dollars. For this reason,it is considered as a potential agent of bioterrorism and in theEuropean Union (EU) as a quarantine organism (Anonymous 2000: CouncilDirective 2000/29/EC), which is subject to strict prevention and controlmeasures regulated by two European directives (Anonymous 1998, 2006:Council Directive 98/57/EC and Commission Directive 2006/63/EC).

R. solanacearum presents great intraspecific diversity, and hastherefore long been considered a complex of species consisting of fourphylogenetic groups or phylotypes. In 2014, after a taxonomic revision,a reclassification of this complex was proposed (Safni et al., 2014).The phylotypes I and III of R. solanacearum they have been classified asthe new species R. pseudosolanacearum and phylotype IV in the newsubspecies R. syzygii subsp. indonesiensis. Phylotype II retains thename of the species R. solanacearum.

In the text of this document the term “the species formerly known as R.solanacearum” is used to refer to R. solanacearum in studies, data,patents, publications, literature, etc., prior to the taxonomic revisionof Safni et al (2014), regardless of whether the name corresponds or notwith the current classification. Furthermore, the term “R. solanacearum”is used to refer to the species R. solanacearum as described after theaforementioned taxonomic revision, i.e. the species is constitutedsolely by strains of phylotype II.

In Spain, as in most EU countries, there have been outbreaks of thedisease caused by R. solanacearum in several areas, usually in potatoesand in some cases tomatoes. In all cases of outbreaks the eradicationmeasures set out in the relevant legal regulations were implemented.However, this bacterium can survive in the environment, water, soil orother reservoirs (Alvarez et al., 2007, 2008, 2010). Thus, subsequentoutbreaks have been associated with irrigation with surface watercontaminated with this bacteria (Caruso et al., 2005), as these are oneof the main routes of introduction and spread of the pathogen into newareas, which comes through irrigation water. In fact, it described thepresence of R. solanacearum in various waterways both in Spain andvirtually all EU countries. Therefore, the EU Directives prohibitirrigation with water contaminated with this bacteria.

This represents a practical problem for the farmer, as the most affectedcrops are irrigated crops (tomato and potato) and irrigation water is ascarce commodity in Spain and other countries of the Mediterraneanbasin, where the bacteria are also present. Also in the area whereoutbreaks of the disease have been detected, host plants cannot be grownover a period of at least 4 years from the detection of an outbreak.

The large capacity of survival of R. solanacearum in the environment(water, soil or other reservoirs) impedes control. In the past and up tothe present moment, control through farming practices and chemicalcontrol are being used. Thus, in areas of the world where this pathogenis present in the soil, usually in developing countries, growing hostplants is problematic and the most widely used methods of control arefarming techniques with varying results. Resistant cultivars have alsobeen obtained, however, resistance may be unstable (Hartman andElphinstone, 1994).

With respect to control via chemical or physical treatments, generallythey are not effective. The application of copper compounds, antibioticsand soil fumigants have been used without much success (Lopez andBiosca, 2005), and are also expensive and have a great impact on theenvironment. Other chemical treatments such as chlorination or physicaltreatments, such as ultraviolet radiation of water contaminated withbacterial pathogens, have limited effectiveness where particles arepresent in the water, even if very small (Marco-Noales et al., 2008).Furthermore, it has been shown that both of aforementioned disinfectanttreatments can induce various bacteria to enter into the viable butnon-culturable state (VBNC) (Oliver et al., 2005 Santander et al, 2012.)Or cause reversible cell damage (McFeters and LeChevallier, 2000). Thesebacterial cells in the VBNC state or having reversible damage canrecover cultivability and pathogenicity after coming into contact withsusceptible host plants (Santander et al., 2012). These circumstancesdemonstrate the need for alternative methods that preferably destroy thebacterial cells.

An alternative would be biological control using specific bacteriophagesof R. solanacearum. This biocontrol strategy has been successful in thetreatment of other diseases caused by phytopathogenic bacteria (Jones etal., 2007).

In that vein, Japanese patent JP4532959-B2 (publication numberJP2005278513) describes three types of bacteriophages with bacteriolyticactivity on Japanese strains of the species formerly known as R.solanacearum and from 2014 belonging to the new species R.pseudosolanacearum. Type 1, with double-stranded DNA genome (dsDNA) ofapproximately 250 kbp, and types 2 and 3, with a genome ofsingle-stranded DNA (ssDNA) of 4.5 and 6 kbp respectively.Bacteriophages are characterized and distinguished by the size of theirgenomes and their activity against six strains of bacteria (C319, M4S,Ps29, Ps65, Ps72 and Ps74), all of which are sensitive to type 1bacteriophages, while the type 2 only lyses one strain (the C319 strain)and type 3 lyses four of the six strains (M4s, Ps29, PS65 and Ps74) anda fifth in one trial (the C319 strain). Trials with restrictionendonucleases have only been performed with type 1 bacteriophages andshow that its genome (dsDNA) targets PstI and KpnI, since in theresulting restriction profiles several distinct bands are observed.Finally, the use of two of the three types of bacteriophages (types 1and 2) to control the disease caused by species formerly known asRalstonia solanacearum (currently R. pseudosolanacearum) by addition tosoil cultivation where the plant to be protected is growing.

Japanese Patent JP4862154-B2 arises from a limitation of the abovebecause, as is indicated therein, “it is not enough to control theeffect.” In this second patent is included a new type of bacteriophagelytic activity against all strains of the species formerly known as R.solanacearum which were tested (only a total of 15 strains) and performcharacterization, but do not demonstrate their capacity for biocontrol,since no biocontrol tests were performed on plants with this new type ofbacteriophage. In short, the second patent only provides a new type oflytic bacteriophage with a range of hosts that is seemingly larger thanprevious types.

Yamada et al (2007) describe the isolation of four types ofbacteriophages that infect specific strains of the species formerlyknown as R. solanacearum, from 2014 belonging to the new species R.pseudosolanacearum, from soil samples taken in different areas Japan.These authors perform structural characterization including amorphological characterization by electron microscopy virions andmolecular characterization by restriction analysis of the four types ofbacteriophages, and some lytic activity tests with cultured bacteria inPetri dishes. Two types of bacteriophages described are mioviruses, thegenus to which all the bacteriophages infecting the species formerlyknown as R. solanacearum (now R. pseudosolanacearum) belong describedbefore publication of the article by Yamada et al, and the other twotypes are filamentous bacteriophages of the inovirus type. Among otherapplications, the usefulness of the bacteriophages' lytic activity asbiocontrol agents for the eradication of the species formerly known asR. solanacearum (now R. pseudosolanacearum) in contaminated soils andpreventing wilting caused by the bacteria in vegetable crops issuggested, showing preference for bacteriophages capable of infecting awide range of pathogenic strains, although only testing 15 strains ofthis host. The only tests with plants included in the aforementionedarticle referred to observing a plant injected with a strain previouslyinfected with one of the isolated lysogenic bacteriophages, concludingthat filamentous phages were not satisfactory for a disease controleffect. This same test method has been used in other studies of the samegroup also with filamentous bacteriophages of the Inoviridae family [seeAddy et al., 2012 and International Patent Application WO2012/147928],where it is shown that aforementioned method of inoculation, in the caseof lysogenic bacteriophage type ΦRSM, leads to the emergence ofavirulent strains of R. solanacearum that help control the diseasecaused by the virulent forms of aforementioned bacteria. However,besides the fact that the inoculation tests have only been performed in5 plants per strain and without repetition [see material and methodsAddy et al., 2012], the practical application of this test method innursery or in the field is highly questionable because the work requiredto inject each individual plant.

In another article by the same research group (Fujiwara et al., 2011)test results demonstrating utility as biocontrol agents on the speciesformerly known as R. solanacearum (currently R. pseudosolanacearum) areprovided for two types of bacteriophages of the Myoviridae familydescribed by Yamada and collaborators (2007, 2010), ΦRSA1 and ΦRSL1, aswell as the effect of other additional bacteriophage ΦRSB1, with waspreviously isolated (Kawasaki et al., 2009). The latter belongs to thePodoviridae family and, like ΦRSA1, is capable of causing lysis in ahigher number of strains, up to 13 of 15 strains of the species formerlyknown as R. solanacearum (of which at least 13 are currently classifiedas R. pseudosolanacearum) (Yamada et al., 2007; Kawasaki et al, 2009).It is shown that treatment of the bacteria with ΦRSA1, ΦRSB1 and ΦRSL1either individually or in possible combinations, except treatment withΦRSL1 alone, results in a rapid decrease in cell density of the hostbacteria, which is only an initial decrease because is followed by theappearance of resistance visible by OD (optical density) in less than 2days. To avoid such resistance, Fujiwara and colleagues (2011) selectedthe use of miovirus ΦRSL1 individually with respect to othercombinations with ΦRSA1 and ΦRSB1, although it is noteworthy that thisis the lowest bacteriophage lytic potential of the three.

Tests performed on plants described in the article by Fujiwara et al(2011), all done with ΦRSL1, entailed two treatments with a suspensionof aforementioned bacteriophage at high concentrations conducted with aspacing of one month, first to the seed and then to the resultingplants; after two days these plants were individually inoculated withthe bacterial suspension by direct contact to the cut tips of the roots(for 30 seconds) and then transplanted. This method of inoculation, likethe previous used by these authors in other publications and patents, itis very difficult if not impossible to implement in nurseries or incultivated fields. Furthermore, in aforementioned publication thereproducibility of the results is not indicated.

Fujiwara et al (2011) also describe stability tests on ΦRSL1 on twoplants pre-treated with aforementioned bacteriophage and soil in contacttherewith, detecting bacteriophages in the roots and in the rhizospheresoil 4 months after inoculation, although it was not verified whetherthe recovered bacteriophages are of the same type as those inoculated.Also, the effect of temperature on the stability of ΦRSA1, ΦRSB1 andΦRSL1 was tested in presence and absence of soil (in SM buffer,Tris-HCl, NaCl, MgSO₄ and gelatine). The stability was monitored foronly 15 days. At the same temperature, major differences in thestability of the three bacteriophages in the presence of soil wereobserved. Both in the absence and presence of soil, the differences weregreater with increasing temperature but, in the absence of soil, markeddifferences start from 28° C. After 15 days incubation in the absence ofsoil at 50° C., 10% of ΦRSL1 bacteriophages survived, ΦRSB1bacteriophages were not detected after 9 days of incubation, and after 3days of incubation in the case of ΦRSA1.

Thus, the work of the Japanese group which includes Yamada, Addy andFujiwara, show that in some cases and with some bacteriophages thatinfect the species formerly known as R. solanacearum (now R.pseudosolanacearum), can be used as agents for prevention of diseasecaused by the bacteria. Except in one case, the podovirus ΦRSB1, ruledout by these authors for biocontrol (Fujiwara et al., 2011),bacteriophages described and used in aforementioned tests belong tofamilies Myoviridae or Inoviridae. In aforementioned tests, thebacteriophages are applied directly to the soil, seedlings or in rootsor stems of plants. No evidence of the potential effectiveness of othermeans of administration of bacteriophages are given, such as perhapsthrough irrigation water. This is not surprising, because in Japan R.pseudosolanacearum has not been detected in natural watercourses, unlikethe case in many European countries and in some areas of the US wherethis type of water is one of the reservoirs and routes of disseminationof R. solanacearum.

The ability to control R. solanacearum in water by using bacteriophageshas been seen in other studies in some areas of eastern and westernEurope. Thus, the summary of the Georgian patent application GEU20041089suggests neutralization of aforementioned bacteria in plants, soil andwater using a mixture of polyvalent bacteriophages in equal proportion.

The present group of inventors, however, has previously suggested theuse of specific bacteriophages of the species formerly known as R.solanacearum in irrigation water to control bacterial wilt caused byaforementioned pathogen (Alvarez et al., 2006a; Alvarez et al, 2006b).The influence of the conditions of watercourses in the survival of thebacteria have been verified, finding that the presence of nativemicrobiota and temperatures of 24° C. favoured the disappearance withrespect to the temperature of 14° C. and sterile water used as a control(Alvarez et al., 2006a). None of these disclosures provides informationabout the phylotype of the tested strains, so it cannot beaforementioned that these phages could act on the current R.solanacearum, consisting of strains phylotype II.

The group of the present inventors have also reported the isolation ofspecific lytic bacteriophages of the species formerly known as R.solanacearum from rivers in Spain (Alvarez et al., 2006a, Alvarez etal., 2006b), but without specifying the method of isolation and thespecific place of isolation of each. The authors have reported initialdata on the characterization of one of them, saying that it seems toshow lytic activity between 14° C. and 31° C., but not at lowertemperatures (9° C.) or higher temperatures (32-39° C.) even in naturalirrigation water pH ranges from 6.5 to 8.2. The initially characterizedbacteriophage appears to be specific to the species formerly known as R.solanacearum, it shows lytic activity on 30 strains of differentorigins, of which the phylotype has not been disclosed. Thebacteriophage showed lytic activity against other bacterial isolates ofriver water. Aforementioned bacteriophage also results in the reductionof bacterial wilt in tomato plants irrigated with water containingmixtures of bacteriophage and the species formerly known as R.solanacearum.

The data obtained by Alvarez and his colleagues support the possibilityof using bacteriophages to prevent and/or control bacterial wilt, inparticular by addition to irrigation water. However, data released bythe group so far do not identify the specific watercourses from whichthe lytic bacteriophages obtained by them can be isolated. Nor wasspecific data given on any of aforementioned bacteriophages in order tofacilitate the identification via the structural characteristics. Dataon the range of temperatures and pH in which aforementionedbacteriophages are active has only been reported for one, which was alsoclaimed to be able to lyse 30 different strains of the species formerlyknown as R. solanacearum, without specifying the particular strains inwhich it has proven activity.

Therefore, there is still a lack of specific lytic bacteriophages whichhave been established to, individually, be able to reduce bacterial wiltwhen added to irrigation water. For the single bacteriophage on whichthis type of tests have been carried out, reflected in the academicpresentation made by the present group of inventors (Alvarez et al,2006a; Alvarez et al, 2006b), data have not been disclosed to allowidentification other than by the lytic activity at different pHs andtemperatures and the claim that decreases bacterial wilt in tomatoplants, not revealing the genus or family or any concrete data onwatercourses in which it is present (and from which it could beisolated) or the specific method of the isolation or the phylotype ofthe host strains that are affected.

Thus, neither for bacteriophages isolated by Alvarez et al. or for thebacteriophages whose use is suggested in the Georgian patent applicationGEU20041089 in the form of polyvalent mixtures, are there available dataon survival under natural conditions, in particular on their survival inthe conditions of the waters in which they would be used. A centralfactor in determining the suitability of a bacteriophage as biocontrolagent is precisely the survival under natural conditions. Whenbacteriophages of phytopathogenic are applied to soil or plants toeliminate aforementioned pathogenic bacteria, the time they can stayactive until they find their target cell is the limiting factor in theprocess, which causes repeated applications of these bacteriophages tobe required for effective control. And in the case of the speciesformerly known as R. solanacearum, its control in watercourses,especially in irrigation water and containers (tanks, storage vessels,reservoirs . . . ) it is important, especially in Europe, because:

i) the main crops affected by R. solanacearum are irrigated (especiallypotato and tomato),

ii) there is currently a shortage of water in Spain and other countriesof the Mediterranean basin where the pathogen is present,

iii) there are official prohibitions throughout the EU to usecontaminated water for irrigation of host plants (Anonymous, 1998, 2006water: Council Directive 98/57/EC and Commission Directive 2006/63/EC),

iv) no control methods available for use in water,

v) a priority objective of EU policy is the conservation of theenvironment (Montesinos et al, 2008; Horizon 2020 Program).

Therefore, it would be desirable to control the populations of bacteriain river water and/or irrigation in a biological treatment that iseffective and respectful of the natural environment. And, as wasaforementioned previously, it is preferable for the method to result inthe death of the bacteria.

In the case of plant pathogen R. solanacearum, the habitats of which arethe host plants and soil, a biological agent that is supplied by watermust have biological characteristics that allow it to survive in thatenvironment, which is not the usual environment of the bacteria or itsspecific bacteriophages. If such survival is be prolonged andbacteriophages maintain their lytic activity on the host after longperiods in water, this further favours applicability in the field asthey can be transferred directly by natural and simple means such aswater, without needing to encapsulated them or to provide other physicaland/or biological means to protect their viability until contact withthe target cell. This high survival rate would also facilitate thepreparation of a commercial form, which could be in an aqueous mediumwithout requiring refrigeration (or even lower temperatures) to maintaineffectiveness.

However, it should be noted that bacteriophages are obligateintracellular parasites, and as such, require the host cell forperpetuation. Since they reach the cell in different ways, depending onthe types of bacteriophages and types of host cells, survival time inthe environment is expected in order to allow them to come into contactwith the host cell. It is known that this time can vary betweendifferent bacteriophages, which requires study in each particular case.For example, significant variations are observed in the survival ofbacteriophages the same serotype/genotype (Brion et al., 2002) or evenbetween bacteriophages of aquatic pathogenic fish bacteria, the naturalhabitat of which is water (Pereira et al., 2011). In the latter case,three months has come to be regarding as good survival time ofbacteriophages in water, accepting that bacteriophages displayingincreased survival in water are good candidates for the control ofbacterial fish diseases in aquiculture (Pereira et al., 2011).

It should be noted that R. solanacearum is a phytopathogenic bacteriumwhose natural environment is frequently the xylem of plants and soil,but not water. Since it is not an indigenous bacteria to aquaticenvironments, specific bacteriophages are not expected to have a highwater survival rate. In fact, none of the previously mentionedpublications and patents describes the viability and specific lyticactivity of lytic bacteriophages of the species formerly known asRalstonia solanacearum in environmental water in the absence of hostcells.

And yet, it would be beneficial to have specific lytic bacteriophagesagainst R. solanacearum, particularly that show a broad spectrum ofstrains of that species on which they are active and that also have ahigh survival rate in water, preferably of at least a month or more,more preferably several months. This could make possible biologicaltreatment of irrigation water contaminated with R. solanacearum, usingspecific bacteriophages of the pathogen, which could prevent or reducebacterial wilt in polluted areas that may impact susceptible plants.Treatment with these bacteriophages present the usual advantages overchemical treatments, as well as those associated with other treatmentsof biocontrol, especially with bacteriophages:

High specificity for the host cell,

Natural replication only in the host cell,

Harmless to other living beings, including microbiota beneficial to theplants to be protected,

Using biological treatment via irrigation water is easier and cheaperthan chemical control, and does not require protective measures forstaff during application, since it is harmless to humans,

Lower environmental impact, especially compared to copper compounds andantibiotics used in agriculture, for which many pathogens have developedresistance, reducing the effectiveness of such chemical treatments andalso increasing the chemical contamination of soil, plants and fruit,

Less legal use restrictions, can be applied where chemical control isprohibited,

Since they are inert in their extracellular state, bacteriophages can becombined with other control strategies and/or biocontrol to increasedisease control,

Easy, low-cost production because they increase their number in thepresence of target bacteria,

Easy adjustment of the dose of bacteriophages to be used depending onthe concentration of pathogen to be treated.

The present invention provides a solution to the problem of the absenceof bacteriophages that display a broad spectrum of strains of thebacterium on which they are active and that also have a high survivalrate in water, preferably at least one month, or more preferably, of atleast several months.

SUMMARY OF THE INVENTION

The present invention is based on isolation, from river water fromvarious regions of Spain, several bacteriophages capable of lysing thebacteria R. solanacearum, and the results of structural, functional andmolecular characterization, as well as the genomics, from which thefollowing have been established:

They all belong to the same viral species, the a new species belongingto the Podoviridae family,

They are specific to aforementioned bacteria,

They are active on a wide range of strains of R. solanacearum,

They are active at different temperatures, pHs, salinity and aerationand in the presence of light,

They have a high survival rate in water, more than three years, innatural waters of different chemical and pH compositions and atdifferent temperatures, in the absence of the host, maintaining theirlytic capacity after such long periods,

They are able to decrease wilt caused by Ralstonia solanacearum ifplants are irrigated with water containing at least one ofaforementioned bacteriophages.

Tests were able to verify that these features are also shared by thebacteriophage for which a partial functional characterization (lyticactivity in liquid medium at different temperatures and pHs, initialhost range and ability to control wilting in plants) was alreadydescribed in previous work of the present group of inventors (Alvarez etal., 2006a, Alvarez et al., 2006b), without the specific origin, methodof isolation, survivability having been described and without amolecular and genomic characterization having been made that would allowtaxonomic classification, for which until now the family to which itbelongs is unknown.

Their high specificity, the wide range of strains they are active onand, most particularly, their high survival rate in water, makes allthese bacteriophages very suitable agents for biocontrol of R.solanacearum in water in natural watercourses and/or irrigation waterand reservoirs, and for the prevention and/or treatment of wilt producedby aforementioned bacteria in plants. This is because maintaining lyticactivity after long periods favours the use in field crops or ingreenhouses or nurseries or in other situations, to which they can betransferred directly via water, a natural and simple way, withoutencapsulating them or any other physical, chemical and/or biologicalmeans to protect the viability until contact with the target cell. Thus,implementation costs are reduced since the number of applicationsrequired over time is reduced, the long-term efficiency of the productis increased in agricultural systems in which it is intended to be used,and outbreaks of disease caused by R. solanacearum can be prevented andcombated more effectively. Thus, control agents of R. solanacearum basedon these bacteriophages have a greater “added value” than other productsfor farmers and nursery keepers, the major potential consumers ofaforementioned product.

In addition, as it was previously stated, such very high survival ratein water of the bacteriophages of the invention, while maintaining thelytic capacity after long periods in aforementioned medium in theabsence of a host was not expected for a specific bacteriophage of abacterium whose natural environment is not water as it is the case of R.solanacearum, and this ability has not been observed in other specificlytic bacteriophages of aforementioned bacteria, isolated from naturalwatercourses of Spain by the present inventors.

Therefore, in a first aspect, the invention relates to a bacteriophagecapable of lysing cells of Ralstonia solanacearum selected from thegroup of:

a) vRsoP-WF2 (DSM 32039), vRsoP-WM2 (DSM 32040), vRsoP-WR2 (DSM 32041),or

b) a podovirus whose genome has the sequence of SEQ ID NO:1(corresponding to vRsoP-WF2), SEQ ID NO:2 (corresponding to vRsoP-WM2)or SEQ ID NO:3 (corresponding to vRsoP-WR2).

Hereinafter, the term “bacteriophage of the invention” is used to referto any one of these bacteriophages.

In another aspect, the invention relates to a composition comprising atleast one of the bacteriophages of the invention, or combinations ofthem. This composition will be considered a composition of the presentinvention.

In a further aspect, the invention relates to the use of at least one ofthe bacteriophages of the invention, or combinations, to control R.solanacearum in natural watercourses, streams of channelled water,natural water reservoirs, irrigation water and irrigation waterreservoirs, by adding one or more of these bacteriophages to irrigationwater or reservoirs.

In another aspect, related to the previous aspect, the invention relatesto the use of at least one of the bacteriophages of the invention, orcombinations, or compositions of the invention to control R.solanacearum in soil by addition of one or more of the aforementionedbacteriophages or a composition of the invention to aforementioned soilthrough irrigation water with which the soil is watered, or pre-treatedwith the aforementioned bacteriophages or aforementioned composition.

In an additional aspect, the invention relates to a method forpreventing or controlling bacterial wilt caused by Ralstoniasolanacearum in plants, comprising the steps of:

a) adding a composition to the water to be used for watering plants,comprising bacteriophages belonging to at least one of thebacteriophages of the invention, or combinations;

b) watering plants with aforementioned treated water.

As indicated previously, the term “Ralstonia solanacearum,” withoutreference to the previous meaning of this term is used in the inventionto refer to the species R. solanacearum as described after the lasttaxonomic review, i.e. the species constituted by phylotype II strains.Conversely, when the term “species formerly known as R. solanacearum” isused, it refers to the bacteria that were considered to be within theterm R. solanacearum in studies, data, patents, publications,literature, etc., prior to the taxonomic revision of Safni et al.(2014), regardless of whether the name corresponds or not to the currentclassification.

Accordingly, the present invention, i.e. the “method for the preventionand/or biological control of wilt caused by Ralstonia solanacearum, bymeans of the use of bacteriophages useful therefor and compositions”,refers to R. solanacearum as described after taxonomic revision of Safniet al. (2014). The three inventions of other authors mentioned in thisdocument refer to the “species previously known as R. solanacearum”that, in cases where information is available (patent documents,publications, etc.), it mostly refers to strains reclassified as the newspecies R. pseudosolanacearum.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows photographs of the culture medium dishes from the lyticactivity tests on the bacteriophages isolated from river water againstRalstonia solanacearum. Darker areas correspond to areas of lysis and/orisolated plaques, which are bacteriophage breeding areas in thebacterial lawn, which allow the lysis of the bacteria to be observed inthe culture medium, the massive growth of the aforementioned bacteriumbeing seen in whitish and opaque areas.

FIG. 2 shows a photograph of a culture medium dish with bacterial lawnfrom strain IVIA 1602.1 of R. solanacearum on which lysis tests wereperformed. In each quadrant, the bacteriophage contained in thesuspension added to the bacterial lawn is indicated; the location of thecontrol quadrant without bacteriophages (upper left quadrant, markedwith the name of the bacterial strain) is also indicated.

FIG. 3 shows a photograph of the bacteriophages of the present inventionobtained by transmission electron microscopy after negative staining. Itis observed that they present a non-enveloped, polygonal head (40 to 60nm in diameter depending on the bacteriophage) and a short tail.

FIG. 4 is a photograph obtained after subjecting to electrophoresis thesamples in which digestion of the DNA of bacteriophages vRsoP-WF2,vRsoP-WM2 or vRsoP-WR2 (as indicated at the top of the photograph) hadbeen carried out with various restriction enzymes indicated above eachcolumn. The columns at the extreme right and left ends correspond to thepattern of molecular weights (M): λ phage DNA digested with HindIII.

FIG. 5 shows the area in which the sequence corresponding to thevRsoP-WM2 bacteriophage presents an insertion of 468 nucleotides to thesequences corresponding to the bacteriophages vRsoP-WF2 and vRsoP-WR2,as well as areas close to this sequence. The presence of a hyphen in asequence indicates a position where a nucleotide is absent in theaforementioned sequence with respect to one of or both of the othersequences, such absence allowing continued alignment in the same area.In the lower line below the corresponding sequence lines, the presenceof an asterisk indicates coincidence between the nucleotides situated atthat position in the three sequences.

FIG. 6 shows the genomic organization of bacteriophages vRsoP-WF2,vRsoP-WM2, and vRsoP-WR2 as compared to bacteriophage T7. In variousshades of grey or with weaves of parallel lines in different directions,the location of functional open reading frames (ORFs) that have beenidentified using the BLAST tool is indicated, as specified in thelegends in the lower part of the figure. It is noted that the threebacteriophages possess a genomic organization and expression of the ORFssimilar to bacteriophage T7 in part of the genome.

FIG. 7 shows the survival curves of bacteriophages vRsoP-WF2, vRsoP-WM2and vRsoP-WR2, at incubated at 14° C. in the absence of host cells inwater from the Tormes river (panel A, top) and the Turia River (panel B,bottom). Survival is expressed as the base 10 logarithm of the plaqueforming units detected per millilitre (PFU/ml) in samples taken at thetimes indicated on the x/y graph.

FIG. 8 shows a graph of the lytic activity of the bacteriophagevRsoP-WF2 added to an initial concentration of 10³ plaque forming unitsper millilitre (PFU/ml) in sterile river water, to which 10⁶ colonyforming units per millilitre (CFU/ml) of Ralstonia solanacearum wereadded. A decrease within time of the CFU/ml corresponding to thebacteria (expressed in the form of the base 10 logarithm, pointsindicated with a filled circle) and an increase in PFU/ml correspondingto the bacteriophage (also expressed as the base 10 logarithm, pointsindicated with a filled square) were observed.

FIG. 9 shows an illustrating scheme of the experimental procedure of theuse of the bacteriophages of the invention on irrigation water developedby the present inventors for the ability to control bacterial wilt. Atthe bottom, photographs of the condition of the plants at the beginningof the test (time zero, top row of photographs) and after 1 month (1month, bottom row of photographs) are shown for each of the combinationsof R. solanacearum and the bacteriophage vRsoP-WF2 indicated.

FIG. 10 shows a bar graph in which reduction of bacterial wilt,expressed as a percentage, in two different trials in tomato plants areshown. Experiment (Exp.) 1, bacteriophage concentration: 10⁹ plaqueforming units per millilitre (PFU/ml) and Exp. 2 bacteriophageconcentration: 10⁶ PFU/ml. In both experiments, the concentration ofbacteria was 10⁵ colony forming units per millilitre (CFU/ml). Verticalframe bars correspond to those plants treated only with bacteria,without bacteriophages; bars without weaving correspond to plantstreated with bacteria and bacteriophage at the indicated concentrations;bars with horizontal weaving correspond to plants treated with bacteriaand 1/10 dilutions of the aforementioned concentrations ofbacteriophages.

FIG. 11 shows a bar graph that represents reduction of bacterial wiltcaused by Ralstonia solanacearum, expressed as a percentage, in trialswhere tomato plants were watered with water containing the combinationsof bacteria (RsoI) and bacteriophage indicated under the bars. The fourcases located further to the right correspond to irrigation water withbinary combinations (from left to right, vRsoP-WF2 with vRsoP-WM2,vRsoP-WF2 with vRsoP-WR2, or vRsoP-WM2 with vRsoP-WR2) or tertiarycombinations (vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2) of bacteriophages withthe bacteria.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned hereinbefore, the invention relates to novel specificbacteriophages of Ralstonia solanacearum (bacteriophages of theinvention), the use of at least one of the bacteriophages of theinvention, or combinations, for R. solanacearum control in naturalwatercourses, natural water reservoirs, irrigation water and irrigationwater reservoirs, by adding one or more of these bacteriophages to theaforementioned water or reservoirs; also it refers to the use of thesebacteriophages to control R. solanacearum in soil, by adding one or moreof these bacteriophages to the aforementioned soil via treatedirrigation water; and a method for preventing or controlling bacterialwilt caused by Ralstonia solanacearum in plants, a composition is addedto water to be used for watering plants, the aforementioned compositioncomprising at least one of the bacteriophages known as vRsoP-WF2 (DSM32039), vRsoP-WM2 (DSM 32040) or vRsoP-WR2 (DSM 32041), or combinations,and the aforementioned plants are watered with the aforementionedtreated water.

In the present application, the word “phage” is used as an abbreviationfor the word “bacteriophage” with the same meaning. Therefore,hereinafter the two terms will be used interchangeably. “Bacteriophage”refers to a virus capable of infecting bacteria, either by producinglysis (lytic cycle) or by inserting itself into the genome andreplicating itself therewith without causing lysis (lysogenic cycle).

The bacteriophages of the invention have been isolated from river waterfrom various regions of Spain, specifically Badajoz, Salamanca and theAlpujarras (Granada).

Morphological characterization by electron microscopy and molecularcharacterization by DNA restriction analysis have shown that the virionsbelong to the Podoviridae family (specifically, the genus of T7-likeviruses), a family of which so far only one bacteriophage has beendescribed with lytic activity against the species formerly known asRalstonia solanacearum, i.e. the bacteriophage ΦRSB1, described byFujiwara et al (Fujiwara et al., 2011) and which has a larger genomethan the bacteriophages of the present invention, whose genome does notexceed 41,000 base pairs (bp) in any of the three cases (see Table 2),while the ΦRSB1 genome has a size of 43,077 bp. Furthermore, the threeinventions of other authors mentioned in the present application,relating to the use of bacteriophages to control R. solanacearum referto the species formerly known as Ralstonia solanacearum, and, whereinformation is available (patent documents, scientific publications,etc.), it can be confirmed that the strains dealt with are mostlyreclassified as the new species R. pseudosolanacearum.

Therefore, the bacteriophages of the present invention do not belong toone of the most common families of bacteriophages lytic for the speciesformerly known as R. solanacearum, i.e. Myoviridae, but rather to adifferent family. In addition, they appear to be part of the samespecies, different from other species of T7-like viruses described sofar. The discovery of viruses belonging to a new species ofbacteriophages which attack R. solanacearum is an unexpected event.

The genome of any of the three bacteriophages of the present inventionappears to have recognition targets for PstI restriction enzyme, whichdoes not result in digestion of them, which represents a difference withlytic bacteriophages of Japanese Patent JP4532959-B2, wherein they aredigested by the enzyme.

Thus, in the present invention are provided first data to identify thethree bacteriophages of the present invention and distinguish them fromany known bacteriophage, such as the family to which they belong, genus,the assignment of all of them to a single species, the sequence of thegenome and distinctive restriction profile, obtained with severalenzymes after digestion of the genome (see Examples 1 and 2, and FIGS.3, 4, 5 and 6). The isolation method used and a source of each isfurther described. Additionally, for clear definition , the depositnumber issued by the Leibniz-Institut DSMZ-Deutsche Sammlung vonMikro-Organismen and Zellkulturen GmbH is provided, as authority forinternational deposit under the Budapest Treaty for each of thebacteriophages.

In addition to the structural characterization (morphologicalcharacterization of the viral particles and genomic characterization),in the present application the functional characterization(physiological and lytic) of the isolated bacteriophage is alsodescribed. As noted previously, one of the important characteristics fora biocontrol agent to be effective is to be shown to act on a wide rangeof strains. As shown hereinafter in the Examples section of the presentapplication, the data previously described by Alvarez et al (Alvarez etal., 2006a, Alvarez et al., 2006b) were confirmed for a bacteriophagethat was known to have been isolated from a watercourse in Spain thathad not been specifically identified and for which insufficientstructural data had been provided to ascribe them to a family and, muchless, to a genus and specific species. In particular, the lytic capacitywas confirmed for 30 strains that, at the time, were all considered tobelong to the same species, i.e. the species formerly known as R.solanacearum, the phylotype of which was unknown, and the pH andtemperature ranges in which it showed activity: between 14° C. and 31°C., and a pH range of 6.5 to 8.2. These data correspond to thebacteriophage referred to in this application as vRsoP-WF2. It has beenfound that the other two bacteriophages of the present invention,vRsoP-WM2 and vRsoP-WR2 exhibit lytic capacity for the same strains ofRalstonia solanacearum, are active in the same ranges of pH andtemperature, thus expanding the aforementioned characterization to thethree bacteriophages of the present invention. The same results wereobtained with mixtures of two of the bacteriophages (vRsoP-WF2 withvRsoP-WM2, vRsoP-WF2 with vRsoP-WR2, or WM2-vRsoP with vRsoP-WR2) or thecombination of all three (vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2). Thus,mixtures comprising combinations of these bacteriophages as well as thecompositions comprising at least one of the bacteriophages of thepresent invention are a possible embodiment of the compositions of thepresent invention.

The specificity for Ralstonia solanacearum was also confirmed, no lyticactivity being observed with bacteria isolated from river water withwhich tests were conducted, or with strains of other species ofpathogenic plant bacteria. Again, these data are valid both for thethree bacteriophages of the present invention and for combinations.

Therefore, the three bacteriophages separately, as well as combinations,fulfil the desirable characteristics for biological control agents suchas high specificity by the host cell, and not posing a risk to themicrobiota of water, soil or plants; being specific against Ralstoniasolanacearum. Nor they pose a threat to the health of humans, animals orplants, being bacteriophages viruses that only infect bacteria. They areactive also in a pH range compatible with the characteristics ofdifferent watercourses of Spain, and in a range compatible with thecharacteristics temperatures. This supports the use of bacteriophages ofthe present invention, individually or as combinations, and ofcompositions comprising such bacteriophages, to control R. solanacearum,whether in water from natural watercourses such as rivers, streams orcreeks, natural reservoirs of water such as lakes, lagoons, ponds,springs and underground accumulations, artificial water reservoirs anddams, covered storage vessels, tanks, ponds (with or without surfacecovers), wells, irrigation water in general, or reservoirs of irrigationwater as well as the aforementioned natural or artificial reservoirs.

In that sense, the field data collected by the present inventors onnatural waters contaminated with R. solanacearum in different Spanishautonomous communities reveal that in the summer months (when thebacteria in water is detected and prohibits its use for irrigation) thehighest daytime temperatures of these waters are between 13° C. and 17°C. and decrease at night. For example, in Salamanca and Guadalajarasampled water temperatures vary between 14° C. and 4° C. In addition, incountries in central and northern Europe with environmental watercontaminated with R. solanacearum temperatures are lower in the summermonths. Therefore, the range of activity observed for the bacteriophagesof the present invention is compatible with use in natural watercourses,particularly in Spain. So is the pH range of action. However, it is noteasy to add bacteriophages to a river watercourse in sufficient amountto achieve effective control of microorganisms therein, especially inthe particular place where this water will be drawn for irrigation, asbacteriophages will be very diluted and will be carried alongwatercourse so that although their survival time is very high, such usedoes not favour bacteriophages to come into contact with the host cellin the section of watercourse that may be of interest, unlessbacteriophages are used in short watercourses and/or watercourses withreduced flow, such as rivulets, brooks and artificial pipes, especiallythose leading to a reservoir where the water will stay for a longertime. Therefore, it is preferred that use should take place in a naturalor artificial water reservoir, such as lakes, lagoons, ponds, streams,reservoirs, covered vessels, tanks, ponds (with or without surfacecovering) or wells. In them, if contamination with R. solanacearum issuspected, it is easier to estimate the extent of such contamination anddetermine the amount or concentration of bacteriophages to add dependingthereon. In any case, it is preferred that the water in the reservoirshould be maintained at a temperature in the temperature range of 4° C.and 30° C. inclusive, interval in which the bacteriophages of thepresent invention survive prolonged periods, keeping their lyticactivity, both alone and as part of compositions containing at least oneof them. This temperature range also comprises the ambient temperaturesof survival and/or multiplication of the pathogen Ralstoniasolanacearum, i.e. the environmental range of 4° C. and 24° C., so thatthe lytic activity of the bacteriophages of the present invention iseffective at the temperatures that such bacteria presents a real threatof development of disease in crops. Since temperatures approaching 30°C. are not common in reservoirs of environmental water, and taking intoaccount fluctuations in daily and seasonal environmental temperature,conditions where the average water temperature in the reservoir isbetween 4° C. and 24° C. inclusive are preferred.

It should be noted that the tests described in Example 4 of the presentapplication confirm the applicability of bacteriophages of the presentinvention via irrigation water and the usefulness in reducing damagecaused by R. solanacearum wilting in plants. That is why the presentinvention also provides a method for preventing or controlling wiltcaused by Ralstonia solanacearum in a plant, comprising the steps ofadding to the water be used to water the plant a composition comprisingat least one of the bacteriophages of the present invention, orcombinations, and watering the plant with the aforementioned treatedwater.

The proposal of pre-treatment of irrigation water before use forirrigation is an option not considered in previous studies of Japaneseauthors discussed hereinbefore and is an option of great interest, sincethe main crops affected by R. solanacearum are irrigated crops. Thus,although it is compatible with the invention that plant cultivation maybe carried out in any of the conditions under which cultivation ispossible, a possible embodiment of the invention which may be veryimportant use on plants growing in a field, in a nursery, in agreenhouse or any other type of substrate, or hydroponics, where it canbe easy to plan and implement the method of the invention within theirrigation system and also do so in ways that benefit many plantssimultaneously. Moreover, but perfectly compatible with theaforementioned embodiment, within the possible application to any cropof a species susceptible and/or tolerant to R. solanacearum, oneembodiment of the invention of great interest is that in which the plantis a species belonging to the family of the Solanaceae (Solanaceaefamily) and in particular one in which the plant is selected from amongtomatoes (Solanum lycopersicum), potatoes (the two crops most frequentlyaffected) (Solanum tuberosum), sweet peppers (Capsicum annuum) oraubergines (Solanum melongena). The application of the method of theinvention is perfectly compatible whether the plant is in a growing areadedicated to plants of a single species, or growing areas where thereare plants of different species, usually with specific sections foreach, as is the case of traditional orchards, usually with an irrigationsystem common to them all and a common irrigation water reservoir. Thecharacteristics of the bacteriophages of the present invention allow forindividual application (plant by plant), as is the case with theapplications proposed by Japanese authors and with other biocontrolagents is not necessary, to be unnecessary. Irrigation can be performedby any known system, such as traditional systems of partial or totalflooding, drip irrigation, subsurface irrigation via perforated pipes,by exudation via porous pipes, or spray irrigation.

As discussed above, it is desirable that, prior to irrigation, the waterwhich the composition with one or more bacteriophages of the presentinvention will be added to should be maintained at a temperature in therange of 4° C. and 24° C. which can be considered a usual environmentalrange, although, as bacteriophages of the invention are active up to atemperature of 31° C., this range can be extended to the range of 4° C.and 30° C. inclusive, although the latter value is unusual inenvironmental water reservoirs. As discussed above, conditions where theaverage water temperature in the reservoir is between 4° C. and 24° C.inclusive are considered suitable, given the daily and seasonalfluctuations of ambient temperature.

It is also desirable that the water pH should be in the range of 6.5 to9.0 (both inclusive) to favour the lytic activity of the bacteriophageof the present invention.

For the same reasons discussed above, it is preferable that, prior toirrigation of the plant with water, irrigation water should stay in areservoir, natural or artificial, from the time when the compositioncomprising one or more bacteriophages of the present invention is added;this approach is consistent with the addition of the bacteriophages whenthe water is not necessarily in such reservoir, but it is a watercoursethat feeds or pours into the reservoir, especially when it is a pipe ora natural watercourse with a low flow rate that flows off of a naturalwatercourse with a high flow rate or a large reservoir, natural orartificial, such as a lake or a dam reservoir.

As for the reservoir itself, can be a storage vessel with or withoutsurface coverage, including tank-type or pond-type; it can also be anatural accumulations of water, such as those that occur in theupwelling of certain springs, or artificial, natural or semi-naturalwells as those formed in certain natural cavities, which are accessed byman at a later time.

On the other hand, it is important to note, as previously commented thatone of the key points that determine the effectiveness of bacteriophagesas biocontrol agents is their survival in the environmental conditionsin which they are intended to be applied. In general, survival of thebacteriophages outside the host, as discussed previously, is extremelyvariable and depends on the particular nature of each bacteriophage,being strongly influenced by the surrounding environment, by conditionssuch as pH of the medium, temperature or sunlight (Iriarte et al.,2007). Because sunlight is a factor that often adversely affects thesurvival of bacteriophages under natural conditions, in considering theuse in surface water, it is important for them to survive adequately inthe presence of this factor. In this regard, the bacteriophages of theinvention were isolated from different Spanish watercourses exposed todifferent levels of sunlight, unlike Japanese bacteriophages isolatedfrom soil and plant material. Moreover, as bacteriophages of theinvention were isolated from water samples in which host cells werepresent, the unexpected survival in water in the absence of host cellswas unknown.

For all these reasons, survival of the bacteriophages isolated by thepresent inventors is considered an important factor, nearly a crucialfeature that is an important advantage for use as a biocontrol agent inwater. As described below in Example 3, the aforementioned survival wastested in the absence of the host cell in natural water from two Spanishrivers (the Tormes River in Salamanca and the Turia River in Valencia)of different chemical composition and pH, and at different temperatures.These two types of water show substantial differences in the mainphysico-chemical parameters referenced in the composition: specificallywith the water of the Turia River values were comparatively 100 timeshigher in Mn, 10 times higher in Fe, between 5 and 10 times higher inchlorides and triple in nitrates; with water from the Tormes Rivervalues being approximately 4 times higher in phosphate; the averagevalues of pH were around 8.13 in the water from the Turia River and 7.36in the water from the Tormes river. Temperature ranges of water rangedfrom 3.5° C. to 20.9° C. for the Tormes River and from 11.5° C. to 22.0°C. for the water from the Turia River, i.e. temperature ranges for bothenvironmental waters are within the temperature values used for testingsurvival of the bacteriophages, which were: 4° C., 14° C. and 24° C.,and the pH values were 7.2 for water from the Tormes River and 8.1 forthe water from the Turia River. Of these rivers, the Tormes River iscontaminated with R. solanacearum and the use of the water is prohibitedfor irrigation, while contamination has not been observed in the TuriaRiver so far. For tests of the present invention, the aforementionednatural water was filtered through a 0.22 μn filter and sterilized, sothat survival tests were performed in the absence of the host. Asdemonstrated in Example 3, the three bacteriophages of the presentinvention were active and at high levels of lytic activity for more than5 months, longer than three months considered good survival period forbacteriophages of aquatic bacteria that affected fish and which aretherefore in their natural environment (Pereira et al., 2011). This highsurvival was observed at the three temperatures tested (4° C., 14° C.and 24° C.), which were intended to cover the environmental range ofinterest for use in watercourses and natural reservoirs of water,artificial reservoirs and irrigation water. Subsequently, as mentionedin Example 3, the test was continued, and it was found that, after 3years in natural water, they remain active. This long period of survivalwith maintenance of lytic activity is unexpected and surprising,particularly for a lytic bacteriophage of R. solanacearum because it isnot a native bacteria from aquatic environments, but rather its naturalenvironment is the xylem of plants and often the ground, and it was notexpected that this bacteriophages specific of such bacteria wouldpresent a high survival rate in water outside the host. In fact, in thestudies of Fujiwara et al (Fujiwara et al., 2011), for example,stability was monitored only for 15 days, in which clear differenceswere observed among the stability of the three bacteriophages tested,more pronounced in a buffer than in the presence of soil, with markeddifferences observed in the survivability of the two bacteriophages ofthe Myoviridae family, ΦRSL1 and ΦRSA1.

As discussed previously, survival outside the host varies greatlybetween different bacteriophages, even among those belonging to the sameserotype/genotype (Brion et al., 2002) or even among those who share acommon natural habitat such as water (Pereira et al., 2011), habitat inwhich survival of aquatic bacteriophages of at least three monthspreviously was considered a suitable characteristic for selecting goodcandidates for the control of bacterial fish diseases transmitted viawater. Thus, the survival of the bacteriophages isolated by the presentinventors was not predictable at all, especially considering that noteven the family to which they belong was known and a high survival ratein water was not expected, since the usual habitat of its host areplants and soil, not water.

Therefore, the survival results in the absence of the host arenoteworthy of the bacteriophages of the invention obtained at 24° C.because the previous results of the present inventors in survivalstudies of R. solanacearum in which lytic activity of otherbacteriophages was tested in the presence of the aforementioned hostbacteria indicated that the temperature of 24° C. promotes the rate ofdisappearance of the pathogen with respect to a temperature of 14° C.(Alvarez et al., 2007). However, at a temperature of 14° C., thebacteriophages of the invention maintain lytic activity on the host innatural water even after three years of the absence, and it has beenobserved that, in conditions similar to natural conditions, studies bythe present inventors with other bacteriophages of this pathogen, lysisalso causes a significant reduction of the populations of this bacterium(Alvarez et al., 2007). In addition, 14° C. is a temperature closer tothose recorded in most aquatic habitats where the pathogen has beendetected in Spain and other European countries.

Moreover, it is noteworthy that it is common conserve biocontrol agents,prior to their use, at low temperatures, preferably at 4° C., but theycan also be stored at 14° C., as well as 24° C. Therefore, it isnoteworthy that the bacteriophages of the present invention remainactive and at high levels at all three test temperatures for more than 5months and the survival with lytic activity can be as long as a periodof three years.

Therefore, a particularly novel feature of the bacteriophages of thepresent invention is the survival for more than 5 months in naturalwater in the absence of the host cell. This is an adequate and veryadvantageous feature for a biological control agent, which must havefeatures that enable it to survive in the medium in which it is intendedto be applied, in this case, water.

As a result, it is compatible with the use of the method and use of thepresent invention that the composition containing bacteriophages shouldbe maintained during storage and/or use, preferably at a temperature inthe range from 4° C. to 24° C., inclusive, which can be considered aregular environmental range, however, since bacteriophages of theinvention are active up to 31° C., this range may extend to a range from4° C. to 30° C. inclusive, despite the latter value not being usual inenvironmental water reservoirs. As discussed above, an averagetemperature of the water in the reservoir from 4° C. to 24° C.inclusive, given the daily and seasonal fluctuations of ambienttemperature, is also considered to be a suitable condition. This enablesthe compositions of the present invention to be easily preserved for along time prior to their use in the form of suspensions in which thebacteriophages are in an aqueous vehicle which can be water(environmental, natural, distilled, previously sterilized, or subjectedto another usual treatment for aqueous vehicles) or an aqueous solution(such as sterile saline, phosphate buffered saline, etc.) and ready touse and apply directly where needed. Therefore, the compositions of thepresent invention may comprise any carrier or excipient agronomicallyacceptable, and may be in liquid form, e.g. as an aqueous suspension,which can be prepared in water or in an aqueous solution and/ordilutions. In this way, they can be used to control R. solanacearum andcan be applied with the method of prevention or treatment of wilt causedby the aforementioned bacteria and can be therefore ready for direct usefrom the stored and marketed form.

The high survival rate, with maintained lytic activity on the host, ofthe bacteriophages of the present invention, favours the use in thefield because they can be transferred directly via water, a natural andsimple way, without encapsulating or adding other physical, chemicaland/or biological mediums to protect their viability until coming incontact with the target cell. This facilitates the production process,lowers costs and eliminates the need for complex formulations forimplementation as well as the addition of chemicals to the environment.Thus, the high survival rate of the bacteriophages in water in theabsence of the target cell reduces the implementation costs bydecreasing the number of uses required over time, and increaseslong-term product efficiency in the agricultural systems where they areintended to be applied, and can thus more effectively prevent outbreaksof disease caused by R. solanacearum. All this leads to a product withmore “added value” for farmers and nursery keepers, the major potentialconsumers of the aforementioned product, i.e. the bacteriophages of thepresent invention and/or compositions comprising them.

Moreover, this high survival in natural water while in the extracellularstate facilitates combination with other control strategies (chemicaland/or physical, and even biological) for the same plant pathogen orothers, which may be an additional optional step of the method of thepresent invention. The method of the present invention is alsocompatible with the use of copper compounds, antibiotics and/or soilfumigants, whose application to the soil where plant is growing can alsobe considered an additional optional step of the method of the presentinvention.

The present invention is also compatible with additional use not only anagent of chemical or physical control, but rather as one or moreadditional biological control agents other than any of thebacteriophages of the present invention (other microorganisms such asbacteria, fungi and other bacteriophages, etc.). One possibility is anyof the lytic or lysogenic bacteriophages previously known, which haveactivity against the aforementioned bacteria. For use, the additionalagent may be further comprised in a composition of the presentinvention, or may be applied separately.

Although the preferred form of the compositions of the present inventionis the liquid form, in aqueous medium, especially when applied to waterto control R. solanacearum and/or preventing or reducing bacterial wiltcaused by the aforementioned bacteria in plants which are to beirrigated with the aforementioned water, other forms of the compositionare also compatible with the invention, especially those known to thoseskilled in the art for the conservation of bacteriophages, such as in alyophilized form (which facilitates preservation at room temperature) oras a refrigerated and/or frozen aqueous suspension, preferably from 4°C. to −20° C., and even lower temperatures, such as −20° C. to −80° C.

As already mentioned, the compositions of the present invention maycontain one of three bacteriophages whose isolation and morphologicaland genomic characterization is described in the present application(vRsoP-WF2, vRsoP-WM2 or vRsoP-WR2), or combinations (vRsoP-WF2 andvRsoP-WM2, vRsoP-WF2 and vRsoP-WR2, vRsoP-WM2 and vRsoP-WR2, orvRsoP-WF2, vRsoP-WM2 and vRsoP-WR2). The tests performed and describedin Example 4 suggest that combinations, either two of or all three ofthe bacteriophages of the present invention are more effective than theuse of the bacteriophages separately, so they may be a good choice foruse against Ralstonia solanacearum in water to be treated, andparticularly in water that is to be used for irrigation in order toprevent or reduce wilt caused by this bacteria. In compositions withcombinations of several bacteriophages, each may be at the sameconcentration as in Example 4 of the present application, but differentconcentrations are also compatible with the invention.

Moreover, an advantage to consider of using the combination of two ormore bacteriophages of the present invention is that mixtures preventthe appearance of strains of R. solanacearum that are resistant to thelytic action of any one of them.

Regarding the total concentration of bacteriophages in the compositionsof the present invention, there are no limitations except those imposedfor chemical reasons, resulting in the suspension being saturated andbacteriophages precipitating or settling. However, in practice, this ishighly unlikely. One option is for the total concentration ofbacteriophages of the invention to range between 10⁵ and 10⁹ plaqueforming units per millilitre (PFU/ml), which are concentrations thathave been tested in the Examples section of the present application andwhich can also be a suggested range of concentrations in order to choosethe final concentration of bacteriophages desired to be present in theirrigation water. However, concentrations may be higher or lower thanthose included in that range, with concentrations of 10³ PFU/ml beingable to be maintained and/or used as in section 4.1 of Example 4, oreven lower, as the present inventors have obtained lysis data in liquidmedium with bacteriophages of the present invention at concentrations ofabout 10² PFU/ml. Thus, the range 10² to 10⁹ PFU/ml or 10³ to 10⁹ PFU/mLare also possible concentration ranges of the compositions of thepresent invention or the conditions of action of the bacteriophages ofthe present invention, as well as other upper or lower limits, since thebacteriophages multiply inside the bacteria.

As far as the different bacteriophages of the present invention, underthe envisaged use and/or maintenance conditions, survival data may leadto a preference for vRsoP-WM2, while macrotests carried out on plantsdescribed in Example 4, specifically on tomato plants, can lead to apreference for vRsoP-WR2, because in such tests a greater reduction inbacterial wilt was observed from applying this bacteriophageindividually with respect to the other two bacteriophages of the presentinvention.

The invention will now be explained in more detail by the Examples andFigures described hereinafter.

EXAMPLES Example 1 Origin and Isolation of the Bacteriophages

Lytic bacteriophages against R. solanacearum were isolated from severalrivers of Castilla-Leon, Extremadura and Andalusia, in the vicinity offields affected by bacterial wilt. A selection of these bacteriophageswas purified and their lytic activity was tested in the laboratoryagainst R. solanacearum, as shown in FIG. 1.

Among them, three bacteriophages (vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2),from different origins, were chosen for further characterization.

VRsoP-WF2: isolated from the Tormes River in the vicinity of Salamanca.

VRsoP-WM2: isolated from the Cayo River in the province of Badajoz.

VRsoP-WR2: isolated from the Yator River in the area of the Alpujarras,in the province of Granada.

The three bacteriophages were purified via successive plaque passages ingeneral LPGA medium (yeast extract [5g]-peptone [5 g]-glucose [10g]-agar [20 g], dissolved in distilled water [1 litre]; the glucose issterilized by filtration and subsequently added to the rest of themedium sterilized by autoclaving) with host cells of a standard strainof R. solanacearum (IVIA 1602.1 strain, deposited at the FrenchCollection of Plant-associated Bacteria [CFBP] under CFBP number 4944and at the DSMZ free access collection under the number DSM 100387).This method is also preferred for amplification of any of theaforementioned bacteriophages of the present invention on a solidmedium.

Once purified, the lytic activity, pH and temperature range, and rangeof hosts of them were characterized as described hereinafter.

1.1. Temperature Range.

The lytic activity observed of the characterized bacteriophages againstthe selected strain of R. solanacearum (IVIA-1602.1) is observed between14° C. and 31° C. in all three cases.

Within this range, the routine incubation temperature of thebacteriophages to multiply in the host, both on solid medium and inliquid medium, under laboratory conditions, is between 28-30° C.,because they are the values that are considered optimal for the growthof R. solanacearum in these conditions.

Mixtures of two (vRsoP-WF2-vRsoP-WM2, vRsoP-WF2-vRsoP-WR2,vRsoP-WM2-vRsoP-WR2) or all three bacteriophages(vRsoP-WF2-vRsoP-WM2-vRsoP-WR2) were also tested. Each of the fourmixtures of bacteriophages also showed activity in the same temperaturerange.

1.2. pH Range.

The lytic activity observed of the characterized bacteriophages againstthe selected strain of R. solanacearum (IVIA-1602.1) tested in differentirrigation water, river water, canal water and lake water, was positivein all of the aforementioned types of water at pHs ranging between 6.5and 9.0. Similarly, the mixtures of bacteriophages showed lytic activityin the same irrigation water and, thus, within the same pH range. Theminimum and maximum pH values for the lytic activity of thebacteriophages have not yet been determined.

1.3. Salinity

Since it has been reported that R. solanacearum can grow in the presenceof NaCl at concentrations of 1%, and even sometimes up to 2%, the lyticactivity of bacteriophages in brackish water of different origins wastested, with salt concentrations of about 1.5%. Lytic activity wasobserved with all three bacteriophages. The four mixtures ofbacteriophages of the invention mentioned in point 1.1 also showed lyticactivity in the tested salinity conditions.

1.4. Visible Light

Since visible light can sometimes affect the lytic activity ofbacteriophages, their activity was tested in conditions of light anddarkness. It was observed that after 48 hours of uninterrupted exposureto intense light (approximately 15,000 lux), the lytic activity wassimilar to that observed in dark conditions, so the presence of lightdoes not affect the lytic activity of the bacteriophages of theinvention. Similarly, the different mixtures of tested bacteriophageshad similar lytic activity both in the presence of light and indarkness.

1.5. Aeration

Since R. solanacearum is an aerobic bacteria and typically grows inliquid medium with stirring (aeration), the effect of the absence ofaeration on the lytic activity was determined, as in field conditionsaeration is not always assured (by example in tank storage). Activitywas observed both in the presence and absence of stirring, of the samemagnitude, being faster with aeration. Similarly, the mixtures of thebacteriophages showed lytic activity both with and without aeration.

1.6. Specificity

Specificity was tested in Petri dishes against bacterial lawns of R.solanacearum strains in the LPGA general culture medium, on which twodrops of a suspension of each of the three bacteriophages (FIG. 2) werepoured. The lytic activity was visualized by the appearance of areas ofclearance formed by lysed bacteria in the bacterial lawn where drops ofthe suspensions with bacteriophages were placed (FIG. 2, correspondingto the test on R. solanacearum strain IVIA-1602.1).

According to experimental data, the lytic activity of the characterizedbacteriophages was positive for 35 strains of R. solanacearum ofdifferent origins, hosts and years of isolation (Table 1). Among theaforementioned, 13 are international in scope and/or standard. Theremaining are all strains isolated in Spain, belonging to the collectionof the Valencian Institute of Agricultural Research (IVIA).

TABLE 1 R. solanacearum strains sensitive to the lytic action of thethree bacteriophages of the present invention. STRAIN CODE COUNTRY OFORIGIN HOST YEAR International Strains NCPPB^(a) 1115 United KingdomPotato 1961 (Ex Egypt) NCPPB 1584 Cyprus Potato 1963 NCPPB 2505 SwedenPotato 1972 NCPPB 2797 Sweden Solanum dulcamara 1974 BR 264 UnitedKingdom Solanum dulcamara 1993 Bordeaux 11-47 France Aubergine 1994Nantes 9-46 France Tomato 1994 550 Belgium (Ex Turkey) Potato 1995IPO-1609 Netherlands Potato 1995 Port 448 Portugal Potato 1995 W 12Belgium Potato 1996 WE 4-96 United Kingdom River Water 1996 Tom 1 UnitedKingdom Tomato 1997 Strains from Spain IVIA^(b)-1602.1 Canary IslandsPotato 1996 IVIA-2049.53 Canary Islands Soil 1999 IVIA-2068.58a CanaryIslands Potato 1999 IVIA-2068.61a Canary Islands Potato 1999IVIA-2093.3.1 Canary Islands Potato 1999 IVIA-2093.5T.1a Canary IslandsPotato 1999 IVIA-2128.1b Castile-Leon Potato 1999 IVIA-2128.3aCastile-Leon Potato 1999 IVIA-2167.1a Castile-Leon River Water 1999IVIA-2167.2b Castile-Leon River Water 1999 IVIA-2528.A₁₋₂ Castile-LeonRiver Water 2001 IVIA-2528.A₃.1 Castile-Leon River Water 2001IVIA-2528.54.A₂ Castile-Leon River Water 2001 IVIA-2751.11 ExtremaduraRiver Water 2003 IVIA-2762.1 Extremadura Tomato 2003 IVIA-2762.4Extremadura Tomato 2003 IVIA-3090.1 Andalusia Tomato 2005 IVIA-3090.5Andalusia Tomato 2005 IVIA-3205.A.22 Castile-La Mancha River Water 2006IVIA-3243 Andalusia Tomato 2006 IVIA-3359.9 Castile-La Mancha RiverWater 2007 IVIA-3359.10 Castile-La Mancha River Water 2007 ^(a)NCPPB:National Collection of Plant Pathogenic Bacteria, United Kingdom.^(b)IVIA: Bacteria Collection of the Valencian Institute of AgriculturalResearch, Spain.

Strains from the NCPPB are available in this international collection.The other strains are available in the IVIA collection ofphytopathogenic bacteria.

Specificity was also tested against other species of plant pathogenicbacteria and various bacterial isolates of river water to assess thepossible impact of the isolated bacteriophages on the microbiota ofnatural water.

The lytic activity was negative for the 14 bacterial isolates of riverwater in the tests, which were selected from various water samples andpresented different colonial morphologies from each other and withrespect to the host. The activity was also negative for the 11 testedphytopathogenic bacteria strains belonging to other genera,demonstrating the specificity of the selected bacteriophages againstRalstonia solanacearum. The same results were obtained with the fourpossible mixtures of the aforementioned bacteriophages.

Example 2 Structural Characterization: Morphological and MolecularCharacterization. 2.1. Morphological Characterization.

A study of the morphology of the selected bacteriophages was carried outvia transmission electron microscopy of the viral particles afternegative staining with phosphotungstic acid. It is observed that theypresent the characteristic morphology of the Podoviridae family:polygonal, non-enveloped heads 40 to 60 nm in diameter and short tails(FIG. 3). The bacteriophages of this family are also characterized by agenome of double-stranded DNA, a fact which was confirmed in the testsdescribed below.

2.2. Molecular Characterization. 2.2.1. Extraction of the DNA of theThree Bacteriophages.

Concentrated capsid suspensions were obtained from the three types ofbacteriophages from the corresponding bacterial lysates (filtered andtreated with DNAse and RNAse to degrade the bacterial nucleic acids), bypolyethylene glycol capsid precipitation protocol. After treatment ofthe aforementioned capsids with proteinase K, extraction of genomic DNAwas performed after the addition of phenol, chloroform and isoamylalcohol. After confirming that concentration and purity were adequate,the obtained DNAs were analysed by electrophoresis in agarose gel toverify the integrity as a preliminary step to the restriction analysis(see section 2.2.2) and purification for subsequent sequencing (seesection 2.2.3).

2.2.2. Restriction Analysis of the Genomes of the Three Bacteriophages.

From the obtained genomic DNAs of the three bacteriophages, restrictionanalysis was carried out with various restriction enzymes, chosen togive a banding pattern belonging to T7 genus bacteriophages of thePodoviridae family. These enzymes were KpnI, ScaI, SpeI and XmnI. PstIwas also tested because it is an enzyme used to cut the genome ofbacteriophages of the species formerly known as R. solanacearumdescribed in Japanese Patent JP4532959-B2 (publication numberJP2005278513).

As shown in FIG. 4, the profile bands obtained with these fiverestriction enzymes is apparently the same for the three bacteriophages:complete digestion with XmnI and partial digestion with KpnI, ScaI andSpeI was observed, while no PstI digestion was appreciable. Theseresults indicate the genetic proximity of the three bacteriophages toeach other, and the difference from the bacteriophages of Japanesepatent application JP4532959-B2, whose genomes themselves are cut byPstI.

2.2.3. Mass Sequencing of Genomic DNAs of the Three Bacteriophages andBioinformatic Analysis.

From the resulting genomic DNA belonging to each of the threebacteriophages, we proceeded to the massive sequencing of the nucleotidebases and subsequent bioinformatic analysis and complete annotation ofthe genomic sequences found (SEQ ID NO: 1, corresponding to vRsoP-WF2;SEQ ID NO: 2, corresponding to vRsoP-WM2 and SEQ ID NO: 3, correspondingto vRsoP-WR2). This part was entrusted to the Valgenetics, S.L.(University of Valencia Science Park, Valencia, Spain).

The main findings were as follows:

The assembly of sequences obtained by massive sequencing yielded finalsequences assembled with 100% fidelity, whose sizes are shown in Table2.

TABLE 2 Size of the Genomic Sequences Obtained for Each Bacteriophage.SEQ ID NO: Bacteriophage Number of Base Pairs (bp) 1 vRsoP-WF2 40,409 2vRsoP-WM2 40,861 3 vRsoP-WR2 40,408

The results indicated that each of the majority sequences included inthe samples of SEQ ID NO: 1 (corresponding to vRsoP-WF2), SEQ ID NO: 2(corresponding to vRsoP-WM2) and SEQ ID NO: 3 (corresponding tovRsoP-WR2) is readily identifiable as a complete genome of abacteriophage belonging to the genus of T7-like viruses, which is thetype species of enterobacteria bacteriophage known as T7 (enterobacteriaphage T7), which belongs to the Podoviridae family.

Comparing the genomes of the three bacteriophages, it was showed thatthey were 99% identical over the whole of the genome. However, analysisof these sequences made obvious the presence of small genomicdifferences such as mutations, insertions and deletions distributedthroughout the genome. These differences are higher in the sequence ofSEQ ID NO: 2 (corresponding to vRsoP-WM2) than in the sequences of SEQID NO: 1 (corresponding to vRsoP-WF2) and SEQ ID NO: 3 (corresponding tovRsoP-WR2), which are almost identical. Thus, the sequence of SEQ ID NO:2 contains an insertion of 468 nucleotides compared to the sequences ofSEQ ID NO: 1 and SEQ ID NO: 3. FIG. 5 shows the sequence alignmentextract corresponding to the area of the insertion. Small differencesfound in the nucleotide sequences indicated that bacteriophagesvRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 are different bacteriophages of thesame viral species (Table 3).

TABLE 3 Comparison of the Sequences of the Genomes of BacteriophagesyRsoP-WF2, yRsoP-WM2 and yRsoP-WR2 Compared Pattern Sequence Sequence ofthe Comparison (SEQ ID NO:) (SEQ ID NO:) Coverage* Identity** 1 2 98%99% 1 3 100%  99% 2 3 99% 99% *Homology between sequences of comparedgenomes, expressed as a percentage. **Nucleotides matching within areasof homology of the genomes compared, expressed as a percentage.

Additionally, using the BlastN and Blast2Seq analyses carried out withthe tools accessible to the public via the website of the NationalCenter for Biotechnology Information of the USA(http://www.ncbi.nlm.nih.gov/), it was found that the genomes ofbacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 exhibit some regionswith high identity (about 70%) with bacteriophages: Ralstonia RSB1,Vibrio VP4 and, especially, Rhizobium RHEph01, all of them being T7-likebacteriophages (Table 4). These regions (corresponding to 5-23% of theentire genome of bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2)belong to highly conserved regions.

TABLE 4 Comparison of the Sequences of the Genomes of BacteriophagesvRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 with Several T7-like BacteriophageGenomes. Compared Sequence Genome of the Virus Pattern (SEQ Sequence ofthe Comparison Coverage* ID NO:) (GenBank Access Number) Identity** 1 ΦRalstonia RSB1 (AB597179.1)  2% 84% 1 Φ T7 (NC_001604.1)  5% 67% 1 ΦRhizobium RHEph01 19% 68% (JX483873.1) 1 Φ Vibrio VP4 (NC_007149.1)  5%70% 2 Φ Ralstonia RSB1 (AB597179.1) 15% 66% 2 Φ T7 (NC_001604.1)  5% 67%2 Φ Rhizobium RHEph01 23% 68% (JX483873.1) 2 Φ Vibrio VP4 (NC_007149.1) 4% 70% 3 Φ Ralstonia RSB1 (AB597179.1) 15% 66% 3 Φ T7 (NC_001604.1)  5%67% 3 Φ Rhizobium RHEph01 22% 68% (JX483873.1) 3 Φ Vibrio VP4(NC_007149.1)  2% 70% *Homology between sequences of compared genomes,expressed as a percentage. **Nucleotides matching within areas ofhomology of the genomes compared, expressed as a percentage.

These results reveal that, except in these conserved regions within theT7-like bacteriophages, the genomes of bacteriophages vRsoP-WF2,vRsoP-WM2 and vRsoP-WR2 contain a highly divergent nucleotide sequencewith respect to other bacteriophages deposited in GenBank. Therefore,these high differences in nucleotide sequence guarantee thatbacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2 correspond to a newspecies within the genus of T7-like viruses.

Moreover, the identification of open reading frames (ORF) andcharacteristic features of the bacteriophages revealed that thesequences of the genomes of bacteriophages vRsoP-WF2, vRsoP-WM2 andvRsoP-WR2 have genomic organization and ORF expression that is mutuallysimilar, and similar to T7-like bacteriophages in part of the genome(FIG. 6).

In summary, the three bacteriophages of the present invention are threeisolates of the same viral species, being a new species classified asbelonging to the genus T7 of the Podoviridae family, with organizationvery similar but distinct from the T7 bacteriophages deposited in GenBank (FIG. 6). The novel bacteriophages have different sequences to T7bacteriophages, only resembling in some highly conserved areas, such asthose related to replication and encapsidation.

Example 3 Survival of the Three Bacteriophages in Natural River Water

The survival of the three selected bacteriophages was tested in twodifferent types of river water: Tormes, from Salamanca, and Turia, fromValencia, both in Spain. These two types of water show substantialdifferences in the main physico-chemical parameters analysed of thecomposition: specifically with the water of the Turia River values werecomparatively 100 times higher for Mn, 10 times higher for Fe, between 5and 10 times higher for chlorides and triple for nitrates; with waterfrom the Tormes River, values were approximately 4 times higher inphosphate; the average values of pH were around 8.13 in the water fromthe Turia River and 7.36 in the water from the Tormes river. Temperatureranges of water ranged from 3.5° C. to 20.9° C. for the Tormes River andfrom 11.5° C. to 22.0° C. for the water from the Turia River, i.e.temperature ranges for both types of environmental water are within thetemperature values used for testing survival of the bacteriophages,which were: 4° C., 14° C. and 24° C., and the pH values were 7.2 forwater from the Tormes River and 8.1 for the water from the Turia River.

Surprisingly, it was observed that all the bacteriophages maintainedtheir lytic activity against R. solanacearum after more than five monthsunder these conditions, in the absence of the host since, prior toinoculation, the water had been filtered through a 0.22 μm filter andautoclaved.

FIG. 7 shows graphics for the evolution of plaque forming units permillilitre (PFU/ml) of the bacteriophages in both the water from theTormes River (panel A) and from the Turia River (panel B), in samplesincubated at 14° C. It is observed that PFU/ml are maintained in theabsence of Ralstonia solanacearum. The survival curves of the threebacteriophages samples kept at 4° C. and 24° C. were similar.

Thus, it is noteworthy that the three bacteriophages are active and havehigh lytic activity at all three temperatures tested for more than 5months.

In addition, survival and maintenance of the lytic activity of allbacteriophages were confirmed at 4° C. and 14° C. for a period of timeas long as three years. This result is important for conservation withinthis temperature range when such long storage periods are required.

Example 4 Biocontrol of Bacterial Wilt Caused by R. solanacearum 4.1.Ability to Control Bacterial Populations in Natural River Water.

Since the three bacteriophages of the invention had similar lyticactivity at the different tested temperatures and pH values in naturalwater, initially one of them was chosen as a model (bacteriophagevRsoP-WF2) to perform biocontrol tests of bacterial wilt caused by R.solanacearum.

A bacteria-bacteriophage coinoculation test was carried out in sterileriver water, in a closed system controlled in the laboratory, forsimultaneous quantification of the population levels of bothmicroorganisms over time. To do this, the bacteria was inoculated at aconcentration of 10⁶ of colony forming units per millilitre (CFU/ml) inthe liquid medium (sterile river water) and the bacteriophage was addedat a concentration of 10³ plaque forming units per millilitre (PFU/ml).As shown in FIG. 8, it was confirmed that populations of the inoculatedbacteria (reference strain IVIA-1602.1 of R. solanacearum) descendedsubstantially in a few hours due to the lytic activity of the inoculatedbacteriophages (bacteriophage vRsoP-WF2), the aforementioned pathogenicbacteria virtually disappearing after about 10 hours.

4.2. Tests of Biocontrol of Bacterial Wilt in Host Plants: BacteriophagevRsoP-WF2.

The ability of the river water bacteriophage vRsoP-WF2 for biocontrol ofthe disease caused by R. solanacearum was tested in two independentexperiments, watering plants of a susceptible host (tomato plants) witha concentration of the standard bacterial strain IVIA 1602.1 (10⁵CFU/ml) and two different concentrations of the aforementionedbacteriophage (10⁶ and 10⁹ PFU/ml), and the decimal dilutions (10⁵ and10³ PFU/ml, respectively), in conditions of optimum temperature andhumidity for the development of the disease. The experimental procedureis shown in FIG. 9.

The results of both experiences are shown in FIG. 10. Overall, thedisease incidence decreased to 0-5% in plants irrigated with thepathogen and the bacteriophage, in independent experiments, while in thecontrols without bacteriophages wilt incidence was 25-50%.

4.3. Tests of Biocontrol of Bacterial Wilt in Host Plants: vRsoP-WF2,vRsoP-WM2, vRsoP-WR2, and their Combinations.

Similar to the experiments of biocontrol in plants carried out with thebacteriophage vRsoP-WF2 described in section 4.2, a macrotest wascarried out, which could simultaneously study the ability for biocontrolof each of the three bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2,separately as well as in combinations of two and a mixture of all three.This is considered a macroassay, since it is performed with a largenumber of plants requiring sufficient room for incubation and peoplequalified for carrying it out. In all cases, tomato plants, which are ahost susceptible to the pathogen, were tested, wherein approximately 35plants per experimental condition were inoculated, which isapproximately 315 plants. Inoculated plants were kept in a climaticchamber of suitable size, in day/night cycles of 16 hours of light at26° C. and 8 hours dark at 22° C. and a humidity of about 70%, inconditions of biological containment, in a BSL3 laboratory. In the citedmacroassay, the concentration of R. solanacearum (strain IVIA 1602.1) inirrigation water was 10⁵ CFU/ml, while the total concentration ofbacteriophages was 10⁷ PFU/ml in all the tested experimental conditions.

The graph of FIG. 11 shows the results obtained. These results indicatethat:

The bacteriophage vRsoP-WR2 is the most effective of the three,resulting in a greater decrease of bacterial wilt when added toirrigation water at the same concentration as the other twobacteriophages.

Any mixture of the bacteriophages (either binary combinations, or thecombination including the three) are more effective than the separatebacteriophages.

All these experiments demonstrate the lytic potential of thebacteriophages of the present invention, isolated at different places inSpain, for the biocontrol of R. solanacearum and, therefore, theapplicability of the aforementioned lytic activity in both the treatmentof environmental water for agricultural use that has been contaminatedwith the aforementioned pathogen, or other uses, such as in theprevention and/or control of the disease caused in the field. Thisbiocontrol ability is especially important, since it is considered thatthere is currently no effective control methods available via soil orwater. And, in this case, as previously discussed and demonstrated inthe aforementioned experiments, the biocontrol agents provided by thepresent invention have the unexpected feature of a high survival rate inwater under normal environmental temperatures in Spain, which it is anadvantage for use on plants via irrigation water and for the control andprevention of the presence of R. solanacearum therein, and for easy andprolonged maintenance of the marketed forms of the bacteriophages of theinvention prior to use. Such maintenance may take place in an aqueousmedium for a long time without severe loss of lytic activity and noteven require, prior to the application to water, pre-dilution of thebacteriophages or their mixtures with any kind of physical or chemicalvehicle to facilitate the interaction with the target bacteria or toensure the stability before coming into contact with it, so thatapplying bacteriophages to irrigation water, water streams or waterreservoirs can be simply by pouring them into R. solanacearumcontaminated water to be controlled.

Deposit of Microorganisms

The bacteriophages vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2, with ability tolyse R. solanacearum cells, have been deposited in the German Collectionof microbial cultures Leibniz-Institut DSMZ-Deutsche Sammlung vonMikro-organismen and Zellkulturen GmbH, Inhoffenstrasse 7B, 38124Braunschweig, Germany, following the rules of the Budapest Treaty forthe deposit of microorganisms for patent purposes, on the followingdates and assigned the following access numbers (Table 5).

TABLE 5 Data on the Deposit of Bacteriophages in the DSMZ GermanCollection. Material Deposit Date Access Number vRsoP-WF2 15 Apr. 2015DSM 32039 vRsoP-WM2 15 Apr. 2015 DSM 32040 vRsoP-WR2 15 Apr. 2015 DSM32041

REFERENCES

-   Addy, H. S., Askora, A., Kawasaki, T, Fujie, M. & Yamada, T. 2012.    Utilization of filamentous phage RSM3 to control bacterial wilt    caused by Ralstonia solanacearum. Plant Diseases. 96:1204-1209.-   Álvarez, B., Biosca, E. G. & Lopez, M. M. 2006a. River water biota    affecting Ralstonia solanacearum survival: characterization of    specific bacteriophages and its potential use for biocontrol in    irrigation water. The 4th International Bacterial Wilt Symposium.    Abst. p. 46. York (UK).-   Álvarez, B., Biosca, E. G. & López, M. M. 2006b. Caracterizacion de    fagos liticos de Ralstonia solanacearum aislados de agua de rio: use    potencial en biocontrol. XIII Congreso de la Sociedad Espa{umlaut    over (n)}ola de Fitopatologia. Abst. p. 62. Murcia-   Álvarez, B., López, M. M. & Biosca, E. G. 2007. Influence of native    microbiota on survival of Ralstonia solanacearum phylotype II in    river water microcosmos. Appl. Environ. Microbiol. 73:7210-7217.-   Álvarez, B., López, M. M. & Biosca, E. G. 2008. Survival strategies    and pathogenicity of Ralstonia solanacearum phylotype II subjected    to prolonged starvation in environmental water microcosms.    Microbiology. 154:3590-3598.-   Álvarez, B., Biosca, E. G. & López, M. M. 2010. On the life of    Ralstonia solanacearum, a destructive bacterial plant pathogen. En:    Current research, technology and education topics in applied    microbiology and microbial biotechnology. Mendez Vilas, A. ed., pp.    267-279. World Scientific Publishing, Singapore.-   Anonymous. 1998. Council Directive 98/57/EC of 20 July 1998 on the    control of Ralstonia solanacearum (Smith) Yabuuchi et al. Off J Eur    Communities L235, 1-39.-   Anonymous. 2000. Council Directive 2000/29/EC of 8 May 2000 on    protective measures against the introduction into the Community of    organisms harmful to plants or plant products and against their    spread within the Community. Off J Eur Communities L169, 1-112.-   Anonymous. 2006. Commission Directive 2006/63/EC of 14 Jul. 2006:    amending Annexes II to VII to Council Directive 98/57/EC on the    control of Ralstonia solanacearum (Smith) Yabuuchi et al. Off Eur    Communities L206, 36-106.-   Brion, G. M., Meschke, J. S. & Sobsey, M. D. 2002. F-specific RNA    coliphages: occurrence, types, and survival in natural waters. Water    Research. 36:2419-2-   Caruso, P., Palomo, J. L., Bertolini, E., Álvarez, B., López, M. M.    & Biosca, E. G. 2005. Seasonal variation of Ralstonia solanacearum    biovar 2 populations in a Spanish river: recovery of stressed cells    at low temperatures. Appl. Environ. Microbiol. 2005. 71:140-8.-   Fujiwara, A., Fujisawa, M., Hamasaki, R., Kawasaki, T., Fujie, M. &    Yamada, T. 2011. Biocontrol of Ralstonia solanacearum by treatment    with lytic bacteriophages. Appl. Environ. Microbiol.    77(12):4155-4162.-   Hartman, G. L. & Elphinstone, J. G. 1994. Advances in the control of    Pseudomonas solanacearum race 1 in major food crops. En: Bacterial    wilt: the disease and its causative agent, Pseudomonas solanacearum.    Hayward, A. C. & Hartman, G. L. eds., pp. 157-177. Wallingford: CAB    International.-   Jones J. B., Jackson, L. E., Balogh, B., Obradovic, A.,    Iriarte, F. B. & Momol, M. T. 2007. Bacteriophages for Plant Disease    Control. Annu. Rev. Phytopathol. 45:245-62.-   Kawasaki, T., Shimizu, M., Satsuma, H., Fujiwara, A., Fujie, M.,    Usami, S. & Yamada T. 2009. Genomic characterization of Ralstonia    solanacearum phage φRSB1, a T7-like wide-host-range phage. J.    Bacteriol. 191:422-427.-   López, M. M. & Biosca, E. G. 2005. Potato bacterial wilt management:    new prospects for an old problem. En: Bacterial wilt disease and the    Ralstonia solanacearum species complex, Allen, C., Prior, P. &    Hayward, A. C. eds., pp. 205-224. APS Press St. Paul, Minn.-   Marco-Noales, E., Bertolini, E., Morente, C. & López M M. 2008.    Integrated approach for detection of nonculturable cells of    Ralstonia solanacearum in asymptomatic Perlargonium spp. cuttings.    Phytopathology 98(8): 949-955.-   McFeters, G. A. & LeChevallier, M. W. 2000. Chemical desinfection    and injury of bacteria in water. En: Nonculturable microorganisms in    the environment. Colwell R. R. & Grimes J. D: eds., pp 255-275.    American Society for Microbiology Press, Washington, DC.-   Montesinos, E., Badosa, E., Bonaterra, A., Penalver, R. & López,    M.M. 2008. Aplicación de la biotecnologia al control biológico de    bacterias y hongos fitopatógenos. En: Herramientas biotecnologicas    en fitopatología. 2008. Pallás, V., Escobar, C.,    Rodriguez-Palenzuela, P. & Marcos J. M. eds., pp. 317-343. Ediciones    Mundi-Prensa.-   Oliver, J D., Dagher, M. & Linden K. 2005. Induction of Escherichia    coli and Salmonella typhimurium into the viable but nonculturable    state following chlorination of wastewater. J Water Health.    3(3):249-57.-   Pereira, C., Silva, Y J., Santos, A L., Cunha, A., Gomex, N. C. M. &    Almeida, A. 2011. Bacteriophages with potential for inactivation of    fish pathogenic bacteria: survival, host specificity and effect on    bacterial community structure. Mar. Drugs. 9:2236-2255;    doi:10.3390/md91112236.-   Safni, I., Cleenwerck, I., De Vos, P., Fegan, M., Sly, L. &    Kappler, U. 2014. Polyphasic taxonomic revision of the Ralstonia    solanacearum species complex: proposal to emend the descriptions of    Ralstonia solanacearum and Ralstonia syzygii and reclassify    current R. syzygii strains as Ralstonia syzygii subsp. syzygii    subsp. nov., R. solanacearum phylotype IV strains as Ralstonia    syzygii subsp. indonesiensis subsp. nov., banana blood disease    bacterium strains as Ralstonia syzygii subsp. celebesensis subsp.    nov. and R. solanacearum phylotype I and III strains as Ralstonia    pseudosolanacearum sp. nov. Int. J. Syst. Evol. Microbiol.    64:3087-3103.-   Santander R. D., Catalá-Senent J. F., Marco-Noales, E. &    Biosca, E. G. 2012. In planta recovery of Erwinia amylovora viable    but nonculturable cells. Trees. 26 (1):75-82.-   Yamada, T., Kawasaki, T., Nagata, S., Fujiwara, A., Usami S. &    Fujie, M. 2007. New bacteriophages that infect the phytopathogen    Ralstonia solanacearum. Microbiology. 153:2630-2639.-   Yamada, T., Satoh, S., Ishikawa, H., Fujiwara, A., Kawasaki, T.,    Fujie, M. & Ogata, H. 2010. A jumbo phage infecting the    phytopathogen Ralstonia solanacearum defines a new lineage of the    Myoviridae family. Virology. 398(1):135-47.

1. A bacteriophage capable of lysing cells of Ralstonia solanacearumselected from the group consisting of a) vRsoP-WF2 (DSM 32039),vRsoP-WM2 (DSM 32040), vRsoP-WR2 (DSM 32041), or b) a podovirus whosegenome has the sequence of SEQ ID NO: 1 (corresponding to vRsoP-WF2),SEQ ID NO: 2 (corresponding to vRsoP-WM2) or SEQ ID NO: 3 (correspondingto vRsoP-WR2).
 2. (canceled)
 3. A composition comprising at least one ofthe bacteriophages of claim 1, or combinations thereof.
 4. Thecomposition of claim 3 comprising one of the following combinations ofbacteriophages: a) vRsoP-WF2 and vRsoP-WM2; b) vRsoP-WF2 and vRsoP-WR2;c) vRsoP-WM2 and vRsoP-WR2; d) vRsoP-WF2, vRsoP-WM2 and vRsoP-WR2. 5.Composition according to claim 4, wherein each of the bacteriophages ofthe combination is present in the same concentration.
 6. (canceled) 7.Composition according to claim 3, wherein the total concentration ofbacteriophages capable of lysing R. solanacearum cells varies between10² and 10⁹ plaque forming units per millilitre (PFU/ml).
 8. (canceled)9. Composition according to claim 3, comprising an agronomicallyacceptable carrier and/or excipient.
 10. Composition according to claim3, further comprising a chemical agent for the control of R.solanacearum or a biocontrol agent of R. solanacearum different from: a)vRsoP-WF2 (DSM 32039), vRsoP-WM2 (DSM 32040), vRsoP-WR2 (DSM 32041), orb) a podovirus whose genome has the sequence of SEQ ID NO: 1(corresponding to vRsoP-WF2), SEQ ID NO: 2 (corresponding to vRsoP-WM2)or SEQ ID NO: 3 (corresponding to vRsoP-WR2).
 11. Composition accordingto claim 3, further comprising a biological control agent of R.solanacearum that is a lytic or lysogenic bacteriophage with activityagainst said bacteria.
 12. Use of a method of using the bacteriophage ofclaim 1 or a composition comprising the bacteriophage, the methodcomprising controlling R. solanacearum in natural watercourses,channelled water streams, natural reservoirs of water, artificial waterreservoirs, irrigation water and irrigation water reservoirs, which willbe used to irrigate crops.
 13. The method according to claim 12, whereinthe bacteriophage of claim or the composition is added to a naturalreservoir of water or to an artificial water reservoir, and wherein thewater in the reservoir is maintained at a temperature between 4° C. and30° C., or the average water temperature in the reservoir is between 4°C. and 24° C., both included.
 14. (canceled)
 15. (canceled)
 16. Themethod according to claim 12, wherein the water pH is in the range of6.5 to 9.0, both included.
 17. Use of a method of using thebacteriophage of claim 1 or a composition for the control of comprisingthe bacteriophage, the method comprising controlling R. solanacearum insoil, by adding one or more bacteriophages or the composition to saidsoil via irrigation water with which the soil is irrigated, which ispreviously treated with one or more aforementioned bacteriophages orwith the aforementioned composition.
 18. A method for preventing ortreating wilt caused by R. solanacearum in a plant, comprising the stepsof: a) adding a composition of claim 3 to the water to be used toirrigate the plant; b) watering the plant with said water.
 19. Methodaccording to claim 18, wherein the water pH is in the range of 6.5 to9.0, inclusive.
 20. Method according to claim 18, wherein the water towhich the composition has been added previously to irrigation, ismaintained at a temperature between 4° C. and 30° C. or an averagetemperature of between 4° C. and 24° C., both included.
 21. (canceled)22. (canceled)
 23. (canceled)
 24. (canceled)
 25. Method according toclaim 18, wherein the irrigation system is a system of partial or totalflooding, drip irrigation, subsurface irrigation via perforated pipes,by exudation via porous pipes, or spray irrigation.
 26. Method accordingto claim 25, wherein irrigation is produced by partial flooding. 27.Method according to claim 18, wherein the plant is growing in a field,in a nursery, in a greenhouse or in hydroponics.
 28. Method according toclaim 18, wherein the plant is a species belonging to the Solanaceaefamily and susceptible to and/or tolerant of R. solanacearum or anyother species susceptible to and/or tolerant of R. solanacearum. 29.Method according to claim 28, wherein the plant is selected from thegroup consisting of potatoes (Solanum tuberosum), tomatoes (Solanurnlycopersicum), sweet peppers (Capsicum annuum), aubergine (Solanummelongena).
 30. Method according to claim 18, further comprising a priorstep to the application of said method in which the irrigation water issubjected to one or more other strategies selected from the groupconsisting of: chemical, physical, and/or biological control for a sameplant pathogen or others.
 31. Method according to claim 18, comprising astep in which copper compounds, antibiotics and/or soil fumigants areapplied to the soil where the plant is growing.